2.4. TFIIH Recruitment and Functions in TC-NER
TC-NER only repairs damage on the transcribed strand of active genes and
is considered more efficient than GG-NER (Hu et al., 2015). A key
difference that distinguishes TC-NER from GG-NER is the presence of
damage-stalled RNA Pol II that serves as the signal for TC-NER
initiation (Lainé & Egly, 2006). The first protein responding to Pol II
stalling is Cockayne syndrome B (CSB), a SWI2-SNF2 type ATPase (Selby &
Sancar, 1997). CSB normally binds to DNA upstream of Pol II to promote
transcription elongation (Kokic et al., 2021; Xu et al., 2017). Upon
transcription stalling, CSB quickly moves to Pol II and functions in
recruiting downstream TC-NER proteins, including Cockayne syndrome A
(CSA) (van der Weegen et al., 2020), a component of an E3 ubiquitin
ligase complex (Groisman et al., 2003). CSA can ubiquitylate CSB as well
as the stalled Pol II (Groisman et al., 2006; Nakazawa et al., 2020).
CSA also physically interacts with UV-stimulated scaffold protein A
(UVSSA) (van der Weegen et al., 2020), another important TC-NER protein.
One mechanism for TFIIH recruitment in TC-NER is through its physical
interaction with UVSSA (Okuda et al., 2017; van der Weegen et al., 2020)
(Figure 3 ). In this regard, it has been shown that UVSSA also
interacts with the PH domain of TFIIH subunit p62 (Okuda et al., 2017),
in a way similar to the interaction between XPC and TFIIH in GG-NER.
Another mechanism for TFIIH recruitment is via Pol II ubiquitylation.
Recruitment of CSA to the stalled Pol II leads to mono-ubiquitylation of
the largest Pol II subunit, Rpb1, at Lys1268 (Nakazawa et al., 2020;
Tufegdžić Vidaković et al., 2020). Interestingly, Rpb1-Lys1268
ubiquitylation enhances the association of the TFIIH core complex with
the stalled Pol II, and this mechanism appears to involve ubiquitylated
UVSSA at Lys414 (Nakazawa et al., 2020). An additional TC-NER factor
that may participate in TFIIH recruitment to stalled Pol II is ELOF1. It
was suggested that ELOF1, a conserved elongation factor, interacts with
both Pol II and the CRL4CSA E3 ligase, and positions
CRL4CSA for Pol II ubiquitylation at the Rpb1-Lys1268
residue(van der Weegen et al., 2021). As Pol II ubiquitylation increases
Pol II-TFIIH interaction (Nakazawa et al., 2020), ELOF1 likely
facilitates this process by enhancing Pol II ubiquitylation.
Despite TFIIH’s roles in DNA unwinding and damage verification in
GG-NER, how TFIIH stimulates TC-NER is much less understood. It is
generally assumed that TFIIH plays identical roles in the two NER
subpathways and there is some evidence supporting this hypothesis. For
example, it has been shown that a helicase-dead XPD mutant abolishes
both subpathways in yeast (Duan et al., 2020). However, it is also
important to note that TC-NER significantly differs from GG-NER in that
the two DNA strands are pre-melted in a transcription bubble by RNA Pol
II (Figure 3 ). When RNA Pol II is stalled by the damage, it is
conceivable that TFIIH may not need to unwind the two strands from
scratch, instead, it is possible that TFIIH may just need to extend the
transcription bubble to ~30 nt for the formation of NER
pre-incision complex. Consistent with this notion, clinical data have
shown that mutations in XPD, the major helicase responsible for DNA
unwinding, are mainly associated with the skin cancer-prone disease,
xeroderma pigmentosum (XP), which is generally considered to be caused
by GG-NER defects (Coin et al., 1998; Lehmann, 2001). Only a small
number of XPD mutations are associated with the severe symptom of XP in
combination with the TC-NER disease, Cockayne syndrome (CS) (Lehmann,
2001; Rapin et al., 2000).
One possible explanation for the clinical observations is that the XPD
mutations in most patients may retain partial helicase activity that is
strong enough to increase the bubble size using the pre-melted DNA in
TC-NER. However, the attenuated helicase activity may not be enough for
generating a repair bubble in GG-NER on an almost fully annealed DNA
double helix. More detailed DNA repair studies in different XPD mutant
cells (e.g., XP-only or XP plus CS) may help us understand the
underlying mechanism for different XPD symptoms and delineate the exact
roles of TFIIH in the two subpathways. Furthermore, to what extent XPD’s
damage verification function is required for TC-NER is also up for
debate; because RNA Pol II stalling should already be a stringent
mechanism to verify the presence of DNA damage. Whether TC-NER needs
both Pol II stalling and TFIIH to verify damage presence needs more
experimental analysis.
It is also still not fully understood if RNA Pol II is evicted from the
DNA to make way for the TFIIH repair complex, along with other NER
factors, or if it simply backtracks along the DNA in the transcription
bubble. There are a number of theories about what could be happening,
but each raises its own questions. If RNA Pol II dissociates from the
DNA, how is it recruited back? Does it retain the transcript in
progress, or does it need to start at the promoter region again? If RNA
Pol II is backtracked, what is the mechanism promoting Pol II
backtracking along the DNA? Considering TFIIH’s DNA helicase function,
future studies should also test a potential role for TFIIH in aiding Pol
II dissociation from DNA or backtracking in TC-NER.